The present invention relates to a cell growth promoter containing domestic silkworm sericin, and particularly to a medical material, a cell culture bed material and a cosmetic material.
Further, the present invention relates to a method for obtaining sericin having a function of growing cells from fibers discharged by domestic silkworms, e.g., cocoon filaments or the like.
Fibers discharged by domestic silkworms, e.g., cocoon filaments or the like, comprise two kinds of proteins, fibroin and sericin. Sericin is present while coat fibroin.
Cocoons are subjected to steps including drying, boiling, thread reeling, etc., followed by degumming (sericin remouing) The silk filaments degummed (fibroin fibers) have been used as silk fabrics.
Further, silk yarn is the only long fiber of the natural fiber and has excellent crystallinity and high strength. Thus, the silk yarn has been used, since old times, also as a thread for operations as well as clothes.
In recent years, silk protein has attracted much attention to its inherent functions and, as a result, has come to be used actively for various applications.
In development of new materials using silk protein, fibroin preceded sericin. In recent years, however, sericin, which is another silk protein, has also become a subject of research and development based on the achievements with respect to fibroin.
For example, there are reports on a cosmetic powder comprising a base the surface of which has been covered with a hydrolysate of sericin (JP-A-10-226626), a cosmetic containing cellulose fiber impregnated with a fibroin dispersion and/or an aqueous sericin solution (JP-A-11-152206), a skin irritation inhibitor containing a sericin hydrolysate as an active component (JP-A-10-245345), a collagen production promoter and a skin preparation for external use for prevention from getting older, both containing a sericin hydrolysate as an active component (JP-A-10-226653), a skin preparation for external use containing a sericin hydrolysate and a degradation product of yeast (JP-A-11-193210) and a cell culture bed comprising a film made up of silk protein(s) (fibroin and/or sericin) of domestic silkworms (Minoura et al., Attachment and growth of cultured fibroblast cells on Silk protein matrices, J. Biomedical Materials Res., Vol.29, 1215-1221(1995)).
On the other hand, when a conventional method in which sericin is recovered from a degumming waste liquor is used as a method for obtaining sericin from cocoon filaments or the like, only products having a low molecular weight such as an average molecular weight of 50,000 or less can be obtained (JP-A-1-168906, JP-A-4-202435, etc.)
In short, since sericin in cocoon filaments are crystallize, it hardly dissolves in water. It, however, has been removed by being degraded in hot alkaline water.
Sericin having a high molecular weight such as an average molecular weight of 100,000 or more has recently become to be obtained efficiently (JP-A-11-92564, JP-A-11-131318, etc.) and a method has been developed in which sericin and the like are eluted from cocoon filaments or the like so that the reduction of molecular weight is controlled (JP-A-11-228837, JP-2000-38514, etc.)
Sericin in domestic silkworms has long been said to be a mixture of proteins having molecular weights of from several tens of thousands to hundreds of thousands. Since it is huge and, therefore, easily forms aggregate or becomes insoluble, it is difficult to handle.
For this reason, the problem that of what types of protein components the fractionation and purification in an undegraded state are constituted has been left unsolved.
Further, in most of the conventional reports in which functions such as a physiological activity are suggested, reduction in molecular weight takes place in an extraction step.
Since we have recently succeeded in extracting and fractionating undegraded sericin, the research of application of these components fractionated has become a main issue.
The object of the present invention is to examine the physiological activities of the components constituting undegraded sericin and to provide new medical materials, cosmetic materials and the like utilizing such functional components.
Means for Solving Problem
For solving the above-described problems, the present inventors first researched the relation between the molecular weight and physiological activity of sericin constituting undegraded sericin. As a result, they obtained information that, of the components constituting undegraded sericin, a sericin having a specific molecular weight has an extremely high cell growth promoting action.
The present invention has been created based on the above information and comprises the following compositions:
Namely, the present invention is present in (1) a cell growth promoter obtainable by elution from a fiber discharged (cocoon filaments or the like) wherein the cell growth promoter comprises sericin having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a main component.
The present invention is present also in (2) a method comprising dissolving sericin contained in a fiber discharged by a domestic silkworm (cocoon filaments or the like) in an aqueous urea solution, and then separating and recovering sericin having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The present invention is present also in (3) the method for separating and recovering according to claim 2, wherein sericin is dissolved in an aqueous urea solution of a concentration of 4 M or more.
Further, the present invention is present also in (4) a cell growth promoter obtainable by dissolving a fiber discharged by a domestic silkworm (cocoon filaments or the like) in an aqueous solution of a neutral salt, wherein the cell growth promoter contains silk fibroin and sericin having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Further, the present invention is present also in (5) a cell growth promoter, wherein the cell growth promoter is in the form of a powder or a film.
Further, the present invention is present also in (6) a cell growth promoter obtainable by degumming a fiber discharged by a domestic silkworm (cocoon filaments or the like) under mild conditions and removing sericin having low molecular weight, wherein the cell growth promoter comprises a fibrous material in a state where the surface of a fibroin filament is covered with a sericin component having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Further, the present invention is present also in (7) a method for producing a fibrous material in a state where the surface of a fibroin filament is covered with a sericin component having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the method comprising degumming a fiber discharged by a domestic silkworm (cocoon filaments or the like) under mild conditions and removing sericin having low molecular weight.
Further, the present invention is present also in (8) a method for producing a fibrous material, wherein scouring by boiling is conducted within mild pH range as close to neutrality as possible.
Further, the present invention is present also in (9) a cell growth promoter obtainable by immersing a liquid silk contained in the middle silk gland and the anterior silk gland in the body of a silkworm, wherein the cell growth promoter comprises sericin having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a substantial main component.
Further, the present invention is present also in (10) a method of using the cell growth promoter described above for a cosmetic material as a skin care material; a wound dressing material, a material for vascular endothelium formation and a material for organ formation as medical materials; and a cell culture bed material as a biological material.
(1) Separation and Identification of Various Types of Sericins Constituting Undegraded Sericin
(a) Obtainment of Undegraded Sericin
Since the use of conventional elution methods using an acidic or alkaline aqueous solution or enzymatic treatment as means for isolating sericin of cocoon filaments or the like results in reduction of molecular weight caused by hydrolysis, these elution methods cannot be adopted.
Therefore, a silk protein solution comprising fibroin and sericin is prepared by dissolving cocoon filaments or the like together with fibroin in an aqueous solution of a neutral salt such as lithium thiocyanate.
By such a dissolution method using an aqueous solution of a neutral salt, a silk protein solution containing approximately undegraded sericin can be obtained with inhibition of molecular weight reduction caused by hydrolysis. The resulting silk protein solution containing undegraded sericin is then subjected to measurement of molecular weight and separation of sericin by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
(Sodium dodecyl sulfate-polyacrylamide gel electrophoresis is hereinafter abbreviated SDS-PAGE.)
As a neutral salt used in the above-mentioned dissolution method using an aqueous solution of a neutral salt, lithium thiocyanate, lithium bromide, calcium chloride, calcium nitrate and the like can be mentioned, and particularly, lithium thiocyanate and lithium bromide are preferred.
Cocoon filaments or the like are added into a concentrated aqueous solution of a neutral salt and dissolved.
During the dissolution, mild conditions are selected for the purpose of inhibiting reduction of the molecular weight of silk protein as much as possible.
The neutral salt is used in a concentration of 50% saturation or more. The heating temperature is set within the range of from room temperature to 80 (xc2x0 C.).
Since reduction of the molecular weight will occur if the temperature exceeds 80 (xc2x0 C.), the temperature is preferably as low as possible considering the dissolution time and the type of the neutral salt.
The dissolution time is determined within the range of from 1 to 20 minutes so that the cocoon filament can be dissolved completely and no reduction of molecular weight occurs in consideration of conditions such as the type of the neutral salt, temperature and the like.
The neutral salt solution containing silk protein dissolved is subjected to dialysis to form a pure aqueous solution of silk protein.
(b) Measurement of Molecular Weight by Electrophoresis
Silk protein is separate by electrophoresis into two components of fibroin (H and L) and four components of sericin (a, b, c and d).
A solution obtained by dissolving cocoon filaments or the like in the aforementioned aqueous neutral salt solution and replacing with a 5 M (5 mol/l) urea solution is treated with a reducing agent, e.g., 2-mercaptoethanol (ME) to break crosslinking bonds (Sxe2x80x94S bonds of proteins).
The resultant is subjected to SDS-PAGE to measure the molecular weight of each protein component constituting cocoon filaments.
The measurement by electrophoresis revealed that sericin a having a molecular weight of about 400,000, sericin d having a molecular weight of about 250,000, sericin b having a molecular weight of about 200,000 and sericin c having a molecular weight of about 35,000 account, respectively, for about 45%, about 34%, about 15.5% and about 5.3% of the cocoon layer sericin.
(2) Preparation of a Material Comprising Sericin a Component
Sericin a component is eluted by heating cocoon filaments or the like in an aqueous urea solution under atmospheric pressure.
The aqueous urea solution has a concentration of 4 M or more, preferably from 7 M to saturation and is kept at from 70 to 100xc2x0 C. for a period of from 3 to 30 minutes preferably from 5 to 30 minutes to extract sericin.
The lower the concentration of the aqueous urea solution is the better, because the lower the concentration of the aqueous urea solution is, the less the amount of the urea and the lower the cost.
However, if the concentration of urea is low, dissolution, in particular, of sericin a is reduced and the amount of sericin a extracted is reduced. Therefore, it is recommended to set the concentration of urea to 4 M or more in order to achieve rapid extraction and a great amount of sericin a recovered.
If the heating temperature is low, it is difficult for sericin to be extracted, whereas a high heating temperature results in degradation of sericin and reduction in molecular weight.
If the extraction time is short, it is difficult for sericin to be extracted, whereas if long, sericin is degraded.
Addition of 1% or more of a reducing agent, e.g., ME, to the aqueous urea solution can increase the recovery rate of sericin.
To the solution of sericin obtained in the manner mentioned above is added an organic solvent, e.g., methanol, ethanol and acetone, in a volume of from 3 to 5 times, thereby obtaining a precipitate of undegraded sericin.
The resultant is dissolved in an aqueous solution of a neutral salt such as lithium thiocyanate containing 1% or more of a reducing agent, e.g., ME or the like, and then organic solvents such as ethanol are added one after another to form a precipitate, which is fractionated into various types of sericins while being checked by SDS-PAGE.
A silk protein having a sericin a content of 90% or more is obtained through repetition of operations comprising dissolution and precipitation of a precipitate.
Alternatively, a silk protein having a sericin a content of 90% or more is obtained by treatment, using a reducing agent (e.g., ME or the like), of a silk protein solution containing fibroin obtained by dissolving cocoon filaments or the like in an aqueous solution of a neutral salt, e.g., lithiumthiocyanate, followed by fractionation of silk protein components by repeating precipitation with an organic solvent such as ethanol and dissolution with an aqueous solution of lithium thiocyanate containing ME.
The sericin a purified through the repetition of dissolution and precipitation is dissolved in an aqueous solution of a neutral salt such as lithium thiocyanate and then desalted by dialysis or the like.
The aqueous solution of the sericin a purified and desalted is spray dried to yield a powdery material.
Further, the aqueous solution of the sericin a purified and desalted is dried on a flat plate to yield a film-like material.
The film-like material may be pulverized into a powdery material.
The above-obtained powdery or film-like material comprising sericin a component can be used, as the entire or a part, as a cell culture bed material, etc. for biological applications, as a wound dressing material, a material for vascular endothelium formation (artificial blood vessel), a material for forming an organ, etc. for medical applications, and as a cosmetic material for a skin care application.
If the powder has an average particle diameter is from 1 to 1,000 xcexcm, preferably from 1 to 100 xcexcm, it can follow the movement of the skin or the like flexibly and, therefore, can be used also as a wound dressing material.
(3) Preparation of a Material Comprising Sericin a and Fibroin
When a material containing sericin a is used especially as a film-like material, it is not necessarily desirable to use a material made up only of sericin a component as received for a wound dressing material or for a medical material, e.g., an artificial blood vessel because of its unsatisfactory strength.
If fibroin, which is another protein component constituting cocoon filaments and is constituted of silk yarn having both good biological compatibility and good strength, is used in combination with sericin a, a material more suitable for medical materials such as a wound dressing material and an artificial vessel can be obtained.
In an embodiment of the use of sericin a and fibroin in combination can be used an aqueous sericin a-fibroin mixed solution obtained by mixing an aqueous solution comprising sericin a and an aqueous fibroin solution prepared by dissolving silk yarn in an aqueous solution of a neutral salt such as a lithium salt.
As the aqueous neutral salt solution, a 5 M or more aqueous solution of lithium thiocyanate, preferably a saturated aqueous solution having a concentration of about 9 M is effective.
The more the amount, relative to a raw silk material, of a solvent, the easier the raw silk material is dissolved, but the more inefficient purification, desalting and the like become.
Thus, if the amount, relative to a raw silk material, of a solvent is reduced as much as possible, silk protein can be recovered efficiently.
For this reason, the amount of the aqueous neutral salt solution to a raw silk material is desirably at least 7 times, preferably from 9 to 50 times the amount of the raw silk material.
The desalting of a solution is carried out by dialysis against water.
Dialysis is carried out until no precipitate is formed from the extradialysis fluid by addition of AgNO3.
Through this operation, an aqueous sericin a-fibroin mixed solution is obtained.
The aqueous mixed solution obtained is dried on a flat plate to form a film-like material.
When forming a film, it is desirable to carry out dialysis as quickly as possible, specifically, within three days, preferably within two days.
If dialysis takes many days, it becomes difficult to form a film because silk protein is caused to gel and become massive.
The aqueous silk protein solution is formed into a film by letting an aqueous silk protein solution flow on a plastic, especially acrylic, plate and drying it quickly so that the crystallinity becomes less than 10%.
For example, it is preferable to dry in a chamber of 60% RH (relative humidity) or less while sending air.
A film having a crystallinity of 10% or more has a poor water absorbability and, therefore, when put on a wound, it cannot absorb an exudate from the wound sufficiently and cannot become flexible. It, therefore, may damage the wound.
For this reason, it is not suitable for a wound dressing material.
A powdery material can be obtained by spray drying an aqueous sericin a-fibroin mixed solution desalted in advance, by freeze drying the aqueous mixed solution, or by precipitating with alcohol or the like, drying and then pulverizing.
It can be obtained also by pulverizing a film-like material.
Since the shape of the powder is different from a film, it can be used as a wound dressing material when the crystallinity of the powder is in the range of from 0 to 100%.
(4) Fibrous Material Comprising a Fibroin Fiber the Surface of which is Covered with Sericin a Component
When a raw silk material such as cocoon filaments, etc. is degummed, sericin d and b, which are easy to be dissolved in water, are removed in a first stage. In a stage where about 40% of sericin has been removed, the surface of cocoon filaments is constituted almost of sericin a and, therefore, it is preferable to use raw silk obtained by completing the degumming at this stage.
When degumming a raw material such as cocoon filaments, etc. by boiling, the farther the pH of the degumming solution is away from neutrality, the easier it is to obtain sericin. However, in this case, since degradation occurs and the molecular weight is reduced, it is preferable to conduct degumming within a mild pH range as close to neutrality as possible.
When degumming is carried out using water, the degumming is carried out by immersing a raw material containing sericin a in water.
In this case, the higher the water temperature, the more efficient it is. To boil water under atmospheric pressure is effective.
For preventing molecular weight reduction, the boiling time is preferably several hours or shorter, more preferably one hour or shorter.
When degumming is carried out using a soap, a soap an aqueous solution of which shows pH of 10 or less, e.g., a marseilles soap having a concentration of 0.3% is used and the boiling is carried out for one hour or shorter, preferably for 30 minutes or shorter.
In materials comprising sericin a and fibroin, the content of sericin a is preferably 5% or more, more preferably 20% or more.
A fibrous material in which the surface of cocoon filaments is covered only with sericin a and which is obtained by degumming a raw silk material such as cocoon filaments or the like by the above-described method and removing sericins d and b, which are easy to be dissolved in water, may be used as medical materials, such as threads for operation, a wound dressing material and an artificial blood vessel, and cosmetic materials, as received or after being processed into woven fabric, nonwoven fabric or the like.
Alternatively, an aqueous silk protein solution is obtained by dissolving and desalting a fibrous material having a surface covered mainly with sericin a, in a method similar to that described in (1)(a) above.
By drying the resulting solution on a flat plate, a film-like material is obtained.
Alternatively, a powdery material is obtained by spray drying of the solution.
Further, a film-like material may be pulverized into a powdery material.
The above-obtained powdery or film-like material comprising sericin a can be used, as the entire or a part, as medical materials such as a wound dressing material and an artificial blood vessel or as a cosmetic material for skin care applications.
If the powder has an average particle diameter is from 1 to 1,000 xcexcm, preferably from 1 to 100 xcexcm, it can follow the movement of the skin or the like flexibly and, therefore, can be used also as a wound dressing material.
In the present invention, the cocoon filament or the like as a raw silk material used for obtaining sericin a (the purity is 90% or more) and a material containing sericin a is defined to be a substance containing cocoon filament sericin discharged by domestic silkworms.
The substance containing cocoon filament sericin includes undegummed materials such as cocoon layers of domestic silkworms, cocoon filaments, raw silk, woven or knitted silk fabrics, their incompletely degummed materials, and fibers, powders and films containing sericin. Of these materials, all of those containing sericin a can be used as a raw silk material.
Further, a cocoon of xe2x80x9csericin silkwormxe2x80x9d which discharges only sericin containing no fibroin can also be used as a raw silk material.
(5) Sericin a from the Middle Silk Gland and the Anterior Silk Gland Liquid Silk in the Body of a Silkworm and a Material Containing Sericin a
The silk gland of a domestic silkworm immediately before discharging silk is removed and is torn in water. The contents, that is, the liquid silk and the silk gland cells, are separated.
The silk gland is separated into the anterior portion, the middle portion and the posterior portion.
In the liquid silk contained in the anterior and middle silk glands, a liquid fibroin several millimeters in diameter is covered with liquid sericin of from 0.1 mm to 0.5 mm thickness.
Since both liquid fibroin and liquid sericin are soluble in water, a mixed aqueous solution containing sericin and fibroin can be obtained by immersing the liquid silk of the anterior and middle silk glands.
Since sericin is dissolved quicker, an aqueous solution containing much sericin can be obtained through 10 to 30-minute immersion. Prolongation of the immersion time results in increase of the ratio of fibroin.
If a method of spray dry is applied for this aqueous solution, a powder can be obtained.
Alternatively, a powder can be obtained also by freeze dry or precipitation using alcohol or the like, followed by drying and pulverization.
If drying the solution on a flat plate, a film can be obtained. A powder can be obtained by pulverizing the film.
(Effect)
The cell growth promoting agent (substance) according to the present invention that contains, as a main component, sericin having a molecular weight, as measured by SDS-PAGE, of about 400,000 obtained by being eluted from fibers discharged by domestic silkworms (e.g., cocoon filaments or the like) is a substance obtained by being eluted from the cocoon filaments or the like while being left almost undegraded. The cell growth promoter is a substantial active component for a cell growth promoting action that sericin constituting cocoon filaments or the like has.
Further, the cell growth promoting agent (substance) that contains, as a main component, sericin having a molecular weight, as measured by SDS-PAGE, of about 400,000 can also be obtained from a liquid silk.
The cell growth promoting agent (substance) has an action of promoting the growth of cells and, therefore, is extremely useful when used in a wound dressing material, a material for vascular endothelium formation and a material for forming an organ for medical applications, in a cell culture bed material for biological applications and a cosmetic material for skin care applications.